Construction of Yeast Two-hybrid cDNA Library on Tomato Spotted Wilt Tospovirus-infected Frankliniella occidentalis
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摘要:
目的 探明番茄斑萎病毒（Tomato spotted wilt tospovirus，TSWV）和介体西花蓟马（Frankliniella occidentalis）之间的分子互作关系，探索介体因子如何参与病毒侵染过程，以期为解析西花蓟马蛋白调控介体传毒机理奠定基础。 方法 通过若虫期饲毒获得高带毒西花蓟马群体，借助SMART技术构建TSWV侵染的西花蓟马酵母双杂交三框cDNA文库。 结果 3种读码框初级cDNA文库的库容量分别为3.0×106、2.0×106 和2.0×106 cfu，文库实际扩增基数大于1.5×106 cfu（3种读码框均大于5×105）。扩增文库平均插入片段主要分布在0.5～3.0 kb。对文库随机挑选16个克隆，测序后与GenBank数据库比对，结果显示各插入片段具有同源序列。 结论 构建的cDNA文库具有较高的库容量和重组率，可用于后续筛选西花蓟马与TSWV互作的蛋白。 -
关键词:
- 番茄斑萎病毒 /
- 西花蓟马 /
- 酵母双杂交cDNA文库
Abstract:Objective A two-hybrid eDNA library was constructed for studying the interaction between tomato spotted wilt tospovirus (TSWV) and Frankliniella occidentalis to decipher the mechanism of the disease transmission by thrip and the factors involved in the virus infection. Method A highly infected F. occidentalis population was created by inoculating TSWV into the thrip nymphs. Thereby, a yeast two-hybrid three-frame cDNA library on TSWV-infected F. occidentalis was constructed using the SMART technology. Result The established 3 reading frame cDNA libraries had the capacities of 3.0×106, 2.0×106, and 2.0×106 cfu. The actual amplification base of the library was more than 1.5×106 cfu or 5 ×105 cfu each with an average insert fragment of 0.5–3.0 kb in length. From the library 16 clones were randomly selected, sequenced, and compared with the GenBank database for of homology verification on the insert fragments. Conclusion The established cDNA library had an ample capacity and high recombination rate adequate for studies on the interactions between TSWV and F. occidentalis. -
图 1 西花蓟马带毒率检测
M：DNA marker DL5 000；1–10：饲毒西花蓟马；CK：健康西花蓟马。
Figure 1. Infection rates of TSWV in F. occidentalis
M: DNA marker DL5 000; 1–10: F. occidentalis feeding on TSWV-infected plants; CK: F. occidentalis feeding on healthy plants.
图 2 携带TSWV西花蓟马总RNA（A）、合成的双链cDNA（B）纯化的双链cDNA（C）电泳
M: 250 bp DNA Ladder (Dye Plus)；1: 总RNA；2: ds cDNA；3: 纯化后ds cDNA。
Figure 2. Agarose gel electrophoresis of total RNA isolated from TSWV-infected F. occidentalis (A), synthetic double-strand cDNA (B), and purified double-strand cDNA (C)
M: 250 bp DNA ladder marker (Dye Plus); 1: Total RNA; 2: ds cDNA; 3: purified ds cDNA.
图 3 酵母双杂交cDNA文库插入片段的PCR检测
M: 250 bp DNA Ladder (Dye Plus)；1–16：重组子；A：阅读框1；B：阅读框2；C：阅读框3。
Figure 3. PCR on cDNA fragments inserted in yeast two-hybrid cDNA library
M: 250 bp DNA ladder (Dye Plus); 1–16: Recombinants; A: Reading frame 1; B: Reading frame 2; C: Reading frame 3.
图 4 质粒电泳检测
M1：Lambda EcoT14 I digest；M2：250 bp DNA Ladder marker (Dye Plus)；1: 文库质粒。
Figure 4. Agarose gel electrophoresis of plasmid
M1: Lambda EcoT14 I digest; M2: 250 bp DNA ladder marker (Dye Plus); 1: Library plasmid.
表 1 引物信息
Table 1. Information on primers
名称
Names引物序列（5′-3′）
Primer sequences（5′-3′）N-F ATGTCTAAGGTTAAGCTCACTAAGGAAA N-R TTAAGCAAGTTCTGCAAGTTTTGTC pGADT7-F GGAGTACCCATACGACGTACC pGADT7-R TATCTACGATTCATCTGCAGC 
下载: 导出CSV
表 2 16个基因序列分析结果
Table 2. Sequences of 16 genes
序号
No.基因名
Gene names片段长度
Length of fragments/bpGenBank登录号
GenBank No.1 未知 Unknown 1493 XM_026425612 2 小泛素相关调节剂 Small Ubiquitin-Related Modifier 695 XM_026421163 3 类NEDD4-结合蛋白1 NEDD4-Binding Protein 1-like 2998 XM_026417919 4 Ras相关蛋白Rab-32 Ras-related Protein Rab-32 2217 XM_026428517 5 40S 核糖体蛋白 S20 40S Ribosomal Protein S20 534 XM_026422691 6 类电压依赖性阴离子选择性通道 Voltage-Dependent Anion-Selective Channel-like 1392 XM_026416490 7 增殖细胞核抗原 Proliferating Cell Nuclear Antigen 1148 XM_026418051 8 类丝氨酸/苏氨酸蛋白激酶Fray2 Serine/Threonine-Protein Kinase Fray2-like 2491 XM_026431489 9 类热休克 70 kDa 蛋白 14 Heat Shock 70 kDa Protein 14-like 1968 XM_026415955 10 相扑偶联酶UBC9 SUMO-conjugating enzyme UBC9 875 XM_026435799 11 类左旋酶 Levanase-like 2404 XM_026432066 12 类蛋白聚糖4 Proteoglycan 4-like 2246 XM_026434373 13 26S 蛋白酶体非 ATP 酶调节亚基 12 26S Proteasome Non-ATPase Regulatory Subunit 12 1760 XM_026430561 14 胞质Fe-S簇组装因子GI11683 Probable Cytosolic Fe-S Cluster Assembly Factor GI11683 1652 XM_026421405 15 类氨基磷酸核糖转移酶 Amidophosphoribosyltransferase-like 2108 XM_026421671 16 动态肌动蛋白亚基4 Dynactin Subunit 4 1853 XM_026420837
下载: 导出CSV
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